ASBTRACT Historically, our understanding of the fundamental properties of hematopoietic stem cell (HSC) and progenitor populations has been largely based on assaying populations of cells. Only recently, single cell transplantation studies have revealed intrinsic heterogeneity among seemingly pure populations of HSCs. However, because of the lack of available tools, the existence and mechanistic dissection of functional heterogeneity in the unperturbed `non-transplanted' marrow has been unfeasible. This is a crucially important question for the hematology field, as how stem/progenitor clones become activated and how clone size is controlled are highly relevant for our understanding of normal and malignant hematopoietic processes. We have recently developed a transposon (Tn)-based system for the simultaneous lineage tracing of thousands of single cells in the unperturbed bone marrow. With this model, we have demonstrated that native non-transplant hematopoiesis is highly polyclonal and mainly driven by the action of cells previously defined as short-term multipotent progenitors (MPPs), and not by the action of long-term HSCs, as historically thought. Here, in the context of this Program Project, we propose to utilize this system to examine the clonal response to different types of hematopoietic injury, and identify the consequences of this response on clonal lifespan and complexity. Our results will be intersected with findings of the Scadden and Zon groups, performing similar studies using complementary approaches in mice and zebrafish. Moreover, using a newly developed approach, we aim to define molecular signatures, at the single cell level, of the heterogeneity in this response. Our data will be analyzed in the context of findings by the Tenen and Scadden labs, also producing molecular signatures of heterogeneity. Additionally, we will study the clonal dynamics of blood production during oncogenic stress and determine the effects of pathway-specific chemical perturbation on the emergence of clonal dominance (Orkin and Zon). Our comprehensive and integrative studies will shed led on the cellular and molecular mechanisms driving functional heterogeneity during organismal stress. Moreover, these studies will inform efforts to enhance hematopoietic regeneration and prevent pre-malignant clonal hematopoiesis.